Immunohistochemical Analysis of p-AKT

The 4-µm-thick CRC paraffin sections were deparaffinized in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. Then, the slides were heated in 0.01 M, pH 6.0 citrate buffer for about 5 min to unmask antigens and incubated in 3% H₂O₂ to inhibit endogenous peroxidase activity. After antigen retrieval, the slides were incubated with anti-p-Akt antibody (Ser-473) (1:200, #YP0006, immunoway, USA) overnight at 4°C, followed by incubation with the secondary antibody (#PV-6001, ZSGB-BIO, China) and visualization with DAB color development kit (#ZLI-9018, ZSGB-BIO, China). The expression of p-AKT in each tumor sample was quantified using H-score (H-score = % of weak staining × 1 + % of moderate staining × 2 + % of strong staining × 3), which ranges from 0 to 300 (14 (link)).